Cells contain networks of signal transduction proteins that mediate their responses to diverse environmental cues. A major challenge is to understand how information is properly routed through these networks such that a given stimuli yields the proper output response, while avoiding cross talk to interlinked components. Misactivation of signaling responses is associated with diverse diseases, including cancer, autoimmunity and diabetes. An emerging paradigm is that scaffold proteins can play an important role in specifically wiring signaling pathways. Scaffold proteins interact with multiple proteins in a pathway, physically organizing them into a complex that may promote their proper communication and insulate them from communication with improper competing partners. Despite the importance of scaffolds, relatively little is understood about the biophysical mechanisms by which they control cellular information flow. Our overall goal is to understand the general molecular mechanisms by which scaffold proteins control signaling efficiency and specificity. We are focusing primarily on Ste5 -- a canonical scaffold protein which coordinates the yeast mating response MAP kinase pathway, and which can be studied using a battery of powerful structural, biochemical, and genetic methods. We are also extending our studies to the analogous MAPK scaffold from humans, KSR. In prior studies, we have mapped individual interaction domains in the Ste5 scaffold protein, determined structures of these domains, and elucidated how a key scaffold domain is a required co-activator for phosphorylation of the mating MAP kinase Fus3. In the current proposal we will focus on the dynamic changes in the scaffold proteins - we have preliminary evidence indicating that the Ste5 scaffold undergoes regulatory conformational and organizational changes during signaling. Thus Ste5 may not simply function as a static tether, but may act as a smart scaffold that normally exists in a basal, inactive state, but upon receiving proper upstream signals, can shift to an active state that promotes flux through the pathway. This model suggests a paradigm by which scaffold proteins may not just passively determine the connectivity of kinase networks, but can also act as gatekeepers that control when and if information flows through the pathway. This mechanism may explain tight control in this pathway -- why related inputs that activate upstream kinases shared by the mating pathway, do not lead to the mating response. We also propose that this type of general conformation gating mechanism may be shared by the mammalian MAPK scaffold, KSR, in order to tightly control the activation of proliferative programs. Our specific aims are to: Aim 1. Elucidate biochemical and structural mechanism of Ste5 scaffold protein autoinhibition Aim 2. Test role of Ste5 autoinhibition in controlling in vivo signaling specificity and elucidate mechanism of Ste5 activation. Aim 3. Test whether similar autoinhibitory mechanism regulates function of the mammalian MAPK scaffold protein KSR Aim 4. Map the supramolecular organization of Ste5 during mating by super-resolution microscopy PUBLIC HEALTH RELEVANCE: A wide range of human diseases, including cancer, inflammation, and diabetes, are associated with malfunctions in signaling pathways. This proposed research will help us understand how signaling pathways function, and how they can fail in disease. In particular our focus on scaffold proteins that wire together specific signaling pathways, may provide novel strategies for therapy involving drugs that disrupt wiring interactions, rather than individual catalytic signaling domains.